Journal: International Journal of Biological Sciences

Article Title: Immune Cell Infiltration and Kynurenine Pathway Activation Define Early Injury and Progression in Diabetic Nephropathy

doi: 10.7150/ijbs.122164

Figure Lengend Snippet: Characterization of renal endothelial cell (EC) subsets and their association with diabetic nephropathy (DN) progression. A UMAP plot showing clustering renal ECs from the HFD/STZ mouse model into three subsets based on marker gene expression: GPX3 high , ESM1⁺, and CD9 high ECs. B Feature plots showing the expression levels of representative marker genes for each EC subset. C Trajectory analysis indicating a differentiation path from GPX3 high (less differentiated) to CD9 high (terminally differentiated) ECs. D Functional scoring of EC subsets showing CD9 high ECs possess the highest glomerular endothelial identity and vascular permeability, while GPX3 high ECs exhibit the lowest scores. E The proportions of EC subsets in diabetic versus control mice. F KEGG pathway enrichment analysis of differentially expressed genes in GPX3 high ECs, highlighting significant enrichment in metabolic pathways. G The involvement of metabolic genes in transitions between EC subsets. H Identification of SELENOP and SPP1 as soluble factors associated with GPX3 high EC formation. I . Immunofluorescence staining revealed the GPX3 + SPP1 + ECs in peritubular capillaries (arrow) of the kidney of two diabetic mouse models. J-K ELISA quantification of SPP1 levels in serum from (J) HFD/STZ and (K) db/db mice at mid- and late-stage DN (n=4 and 5 respectively), confirming elevated SPP1 in disease states. L Overexpression of GPX3 increased SPP-1 expression in GECs (n=4). * , p < 0.05, ** , p < 0.01, *** , p < 0.001, **** , p < 0.0001, ns, no significant. Expression values are presented as mean ± SD.

Article Snippet: GECs were transfected with control cDNA (Cat SKU PS100010 ) or GPX3 cDNA (Cat SKU MG222219 , Origene) for 48 h, the cell supernatant was collected and the SPP1 level was measured by ELISA.

Techniques: Marker, Gene Expression, Expressing, Functional Assay, Permeability, Control, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Immune Cell Infiltration and Kynurenine Pathway Activation Define Early Injury and Progression in Diabetic Nephropathy

doi: 10.7150/ijbs.122164

Figure Lengend Snippet: Illustration of the mechanism of dynamic immune-inflammatory landscape of DN from early to late stage. CCL4⁺ myeloid-derived suppressor cells (MDSCs) with a pro-inflammatory phenotype emerged as the predominant infiltrating immune population in DN progression, with S100A9 highly expressed in MDSCs. The long non-coding RNA MALAT1 was identified as a key factor in maintaining MDSC function. In the early stage of DN, antigen-presenting cell activity was decreased, while in the late stage of DN, elevated expression of CD9 and TREM2 in kidney macrophages suggested a role for lipid-associated macrophages in DN progression. Endothelial cell reprogramming characterized by GPX3 and SPP1 expression was observed during DN advancement. Furthermore, upregulation of KYNU, a key enzyme in the kynurenine pathway (KP), was detected in proximal tubule injury during early stage of DN, while KP metabolites including tryptophan, kynurenine, 3-hydroxykynurenine, and quinolinic acid have the potential to predict adverse renal outcomes in T2D patients.

Article Snippet: GECs were transfected with control cDNA (Cat SKU PS100010 ) or GPX3 cDNA (Cat SKU MG222219 , Origene) for 48 h, the cell supernatant was collected and the SPP1 level was measured by ELISA.

Techniques: Derivative Assay, Activity Assay, Expressing

Journal: Chemical research in toxicology

Article Title: ATF3 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an N 6 ‑Methyladenosine-Based Epitranscriptomic Mechanism

doi: 10.1021/acs.chemrestox.1c00206

Figure Lengend Snippet: ATF3 transcriptionally regulates ABCB1 in TamR MCF-7 cells. (A) Western blot images and (B) quantification results for examining the levels of ABCB1 protein in MCF-7 and TamR MCF-7 cells, TamR MCF-7 cells after knockdown of ATF3, or TamR MCF-7 cells upon the overexpression of YTHDF2 and its W432A mutant. The relative levels of ABCB1 protein were measured from band intensities using ImageJ and are depicted relative to the level detected for control cells. (C) Relative levels of ABCB1 mRNA in parental and TamR MCF-7 cells or in TamR MCF-7 cells after knockdown of ATF3. (D) TamR MCF-7 cells with a stable knockdown of ABCB1 were treated with different concentrations of 4-OHT for 72 h, and cell viability was measured using CCK8. The data were normalized against the control groups (ethanol) and represented as mean ± SD ( n = 3). (E) ChIP-qPCR data showed thw occupancy of ATF3 in the enhancer regions of ABCB1 genes in TamR MCF-7 cells. The p -values were calculated on the basis of unpaired, two-tailed Student’s t -test: *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, p < 0.001; ns, not significant (i.e., p > 0.05).

Article Snippet: Primary antibodies employed for Western blot analysis included those recognizing human ATF3 (Santa Cruz Biotechnology, sc-81189), YTHDF2 (EMD Millipore, Q2490423), ABCB1 (ABclonal, A11747), and AK4 (Santa Cruz Biotechnology, sc-271161).

Techniques: Western Blot, Knockdown, Over Expression, Mutagenesis, Control, ChIP-qPCR, Two Tailed Test

Journal: Viruses

Article Title: Hepatitis C Virus-Induced FUT8 Causes 5-FU Drug Resistance in Human Hepatoma Huh7.5.1 Cells

doi: 10.3390/v11040378

Figure Lengend Snippet: Inhibition of P-gp and MRP1 increased the 5-FU drug sensitivity after HCV infection. ( A , C ) Western blot analysis of P-gp and MRP1 expression in Huh7.5.1 cells in the presence or absence of NSC23925 (P-gp inhibitor) and MK571 (MRP1 inhibitor). Western blot quantification analysis is also listed as ( B , D ). ( E – H ) Analysis of the cytotoxicity of drugs (5-FU/MTX/ADR/CDDP) in HCVcc-infected Huh7.5.1 cells in the presence of P-gp and MRP1 inhibitors.

Article Snippet: P-glycoprotein (P-gp) inhibitor NSC23925 and multidrug resistance-associated protein-1 (MRP1) inhibitor MK571 were purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO).

Techniques: Inhibition, Infection, Western Blot, Expressing

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in MDR1-transfected HEK293 cells. HEK293 cells were stably transfected with empty vector (EV) or full-length human MDR1 plasmids. (A) Protein expression of MDR1 was detected by Western blot analysis. β-actin was used as a loading control. HEK-EV and MDR1 transfected cells were treated with rhodamine 123 (25 μM) or paraquat (100 μM) for 30 min (uptake period), washed, and then incubated in fresh culture medium for 60 min (efflux period). (B) Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) Intracellular paraquat accumulation was quantified by ELISA and normalized to protein lysate concentrations. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with HEK-EV cells.

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Expression and function of MDR1 transporter in RPTEC cells. (A) Protein expression of MDR1 in RPTEC cells was detected by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period) and then treated with the culture media with or without PSC833 (2 μM) for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. Data are presented as mean ± SE (n = 4). Asterisk (*) represents statistically significant differences (p < 0.05) compared with RPTEC cells treated only with rhodamine 123 (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with paraquat (100 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). (A) Intracellular paraquat accumulation was quantified by ELISA normalized to protein concentrations of the cellular lysates. (B) Paraquat concentrations in the culture media were quantified by ELISA and compared with levels detected in RPTEC cell lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated only with paraquat (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following siRNA knockdown of MDR1. RPTEC cells were transfected with siRNA duplexes targeted against human MDR1. (A) Protein expression of MDR1 at 72 h was assessed by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). MDR1 siRNA transfected RPTEC cells were treated with rhodamine (25 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) RPTEC cells were treated as described in (B) using paraquat (100 μM) as a substrate. Intracellular paraquat accumulation was quantified by ELISA and normalized to protein concentrations of the cellular lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells without PSC833 or MDR1 siRNA.

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Cytotoxicity of paraquat in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with different concentrations of paraquat (0–25 mM) for 3 h in the presence and absence of the MDR1 inhibitor PSC833 (2 μM) (uptake period) and then incubated in fresh culture media with or without PSC833 for 69 h (efflux period). (A) Cytotoxicity was assessed using the LDH assay and expressed as percent of LDH released into the cell culture media relative to total LDH activity. (B) RPTEC cells were pretreated with vehicle or PSC833 (2 μM) for 2 h and then treated with vehicle or paraquat (100 μM) for 24 h. Protein expression of Ho-1 was semi-quantified by Western blot. β-actin was used as a loading control. Data are presented as mean ± SE (n = 3). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated by paraquat (no PSC833).

Article Snippet: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

Techniques: Inhibition, Incubation, Lactate Dehydrogenase Assay, Cell Culture, Activity Assay, Expressing, Western Blot, Control